Nkx3.2 fragment and pharmaceutical composition comprising same as active ingredient

ABSTRACT

An Nkx3.2 fragment with improved stability under a histopathological environment of arthritis and a pharmaceutical composition containing the Nkx3.2 as an active ingredient are disclosed. The Nkx3.2 fragment has a function to activate NF-κB at the similar level to full-length Nkx3.2 and resistance to proteolysis by Siah1. In addition, the Nkx3.2 fragment exhibited at least a 10-fold improvement in degenerative arthritis treatment effect compared with Nkx3.2 in an animal model-based in vivo efficacy evaluation. Therefore, the Nkx3.2 fragment can be favorably used in the prevention or treatment of arthritis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 16/348,540, which is a National Stage of International ApplicationNo. PCT/KR2017/012651, filed Nov. 9, 2017, claiming priority to KoreanPatent Application No. 10-2016-0149090, filed Nov. 9, 2016.

TECHNICAL FIELD

The present invention relates to an Nkx3.2 fragment with improvedstability under a pathological tissue environment of arthritis, and apharmaceutical composition comprising the same as an active ingredient.

BACKGROUND ART

Degenerative arthritis, which is one of the most commonly occurringarthritis, is a disease in which degenerative changes damage cartilagetissues that protect a joint, bones and ligaments that form a joint, andthe like, thereby resulting in inflammation and pain. Conventionally,treatment of degenerative arthritis has been carried out primarilythrough control of inflammation. However, it has been proven that thecontrol of inflammation cannot be a fundamental therapeutic technique.

Therefore, in order to treat the cause of degenerative arthritis,identification of a target that regulates processes, such as generation,differentiation, death, calcification, of chondrocytes and developmentof methods to control the target are required.

Meanwhile, overexpressed Nkx3.2 (NK3 homeobox 2) has been shown tosuppress loss of cartilage tissue caused by degenerative arthritis, andthus the protein may be used for treatment of degenerative arthritis. Inthis regard, Korean Patent No. 10-1150900 describes a composition fortreating arthritis, an arthritis diagnostic kit, or a method ofscreening a therapeutic agent for arthritis using Nkx3.2 protein.

In addition, it has been shown that degradation of Nkx3.2 protein ispromoted by the Indian Hedgehog (Ihh) signaling, which is activatedduring the process of hypertrophy and calcification of chondrocytes, andthis phenomenon is mediated by a proteolytic enzyme, Siah1. Furthermore,it has been shown that the Indian Hedgehog signaling increases withdevelopment of degenerative arthritis accompanied by chondrocytecalcification, and controlling the Indian Hedgehog signaling suppressesthe progression of degenerative arthritis in animal models.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present inventors conducted studies to develop therapeutics fordegenerative arthritis using Nkx3.2 variants that can effectivelyfunction under the pathological environment of degenerative arthritis.Consequently, the present inventors produced Nkx3.2 fragments that areresistant to proteolysis induced by Siah1. The present inventors alsoidentified that the aforementioned Nkx3.2 fragments can induce NF-κBactivation at the level comparable to the full-length Nkx3.2.Furthermore, the present inventors found that the Nkx3.2 fragmentsexhibit remarkably improved therapeutic efficacy against degenerativearthritis as compared to the full-length Nkx3.2.

Solution to Problem

In order to achieve the above objects, the present invention provides apolypeptide represented by the following Formula (I):N-terminal extension domain-core domain-C-terminal extensiondomain  (I),

in the above Formula (I),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 1;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 35 in which 1 to 53 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 35; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 5 in which 1 to 23 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

The present invention also provides a polypeptide represented by thefollowing Formula (II):N-terminal extension domain-core domain-C-terminal extensiondomain  (II),

in the above Formula (II),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 37;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 39 in which 1 to 41 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 41 in which 1 to 15 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 16 of SEQ ID NO: 41.

Furthermore, the present invention provides polynucleotides encoding theaforementioned polypeptides.

In addition, the present invention provides expression vectorscomprising the aforementioned polynucleotides.

Furthermore, the present invention provides host cells harboring theaforementioned expression vectors.

In addition, the present invention provides pharmaceutical compositionsfor preventing or treating arthritis, comprising any of theaforementioned polypeptide as an active ingredient.

Furthermore, the present invention provides recombinant virusescomprising the aforementioned polynucleotides.

In addition, the present invention provides pharmaceutical compositionsfor preventing or treating arthritis, comprising any of theaforementioned recombinant virus as an active ingredient.

Furthermore, the present invention provides methods of preventing ortreating arthritis, comprising the step of administering theaforementioned pharmaceutical compositions to a subject in need thereof.

Advantageous Effects of Invention

The Nkx3.2 fragments of the present invention have the function ofactivating NF-κB at the level comparable to the full-length Nkx3.2 andare resistant to proteolysis mediated by Siah1. In addition, theaforementioned Nkx3.2 fragments exhibit improved therapeutic effectsagainst degenerative arthritis as compared with the full length Nkx3.2in animal model-based in vivo efficacy evaluation. Thus, the Nkx3.2fragments can be effectively used for preventing or treating arthritis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is photographic illustration showing the resistance of Nkx3.2fragments against proteolysis mediated by Siah1.

FIG. 2 is photographic illustration showing the binding of Nkx3.2fragments to IκBα.

FIG. 3 is photographic illustration showing induction of degradation ofIκBα by NKx3.2 fragments.

FIG. 4 is a graph showing activation of the transcriptional activity ofNF-κB by NKx3.2 fragments.

FIG. 5 is a schematic diagram depicting the molecular mechanismunderlying the NF-κB activation process induced by Nkx3.2.

FIG. 6 is a schematic diagram for the animal experiment procedure forevaluation of the therapeutic effect of Nkx3.2 fragments using adegenerative arthritis-induced animal model.

FIG. 7 is photographic illustration showing histopathological evaluationof the therapeutic efficacy against degenerative arthritis of Nkx3.2 orNkx3.2 fragments expressed in the affected areas.

FIG. 8 is a graph showing the severity of degenerative arthritis on ascale of 0 to 5 based on quantitative evaluation of overall dataobtained through histological analysis.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention provides a polypeptide represented by thefollowing Formula (I):N-terminal extension domain-core domain-C-terminal extensiondomain  (I),

in the above formula (I)

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 1;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 35 in which 1 to 53 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 35; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 5 in which 1 to 23 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

The core domain is a polypeptide comprising the amino acid sequence fromposition 166 to position 309 of the full-length Nkx3.2 protein. Thefull-length Nkx3.2 protein may include the amino acid sequence of SEQ IDNO: 7, and the core domain may include the amino acid sequence of SEQ IDNO: 1.

The N-terminal extension domain is a domain bound to the N-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 112 to position 165 of the full-lengthNkx3.2 protein. The N-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 35.

The N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 35, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 35 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, or 53 amino acid residues are deleted fromthe N-terminus to C-terminal direction, starting from the amino acid atposition 1 of SEQ ID NO: 35. In some embodiments of the presentinvention, the N-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 35, or a polypeptidecomprising the amino acid sequence of SEQ ID NO: 35 in which 11, 18, 38,41, 44, 47, 50, or 53 amino acid residues are deleted from theN-terminus to the C-terminal direction, starting from the amino acid atposition 1 of SEQ ID NO: 35.

The C-terminal extension domain is a domain bound to the C-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from positions 310 to 333 of the full-length Nkx3.2protein. The C-terminal extension domain may include the amino acidsequence of SEQ ID NO: 5.

The C-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 5, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 5 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acidresidues are deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

Specifically, the C-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5, or a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5 in which 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues are deleted fromthe C-terminus to the N-terminal direction, starting from the amino acidat position 24 of SEQ ID NO: 5.

In some embodiments of the present invention, the C-terminal extensiondomain may be a polypeptide comprising the amino acid sequence of SEQ IDNO: 5, or a polypeptide comprising the amino acid sequence of SEQ ID NO:5 in which 13, 15, 17, 19, 21, or 23 amino acid residues are deletedfrom the C-terminus to the N-terminal direction, starting from the aminoacid at position 24 of SEQ ID NO: 5.

Deletion of the amino acid residues may occur in either or both of theN-terminal extension domain and the C-terminal extension domain. Incertain embodiments, the polypeptide may include the amino acid sequenceof SEQ ID NO: 13, 14, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

The present invention provides a polypeptide comprising the amino acidsequence of SEQ ID NO: 13 or a fragment thereof. The fragment may be apolypeptide comprising the amino acid sequence of SEQ ID NO: 13 in which1 to 53 amino acids are consecutively deleted from the N-terminus to theC-terminal direction, starting from the amino acid at position 1 of SEQID NO: 13. In addition, the fragment may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 13 in which 1 to 23 amino acids areconsecutively deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 333 of SEQ ID NO: 13.

In other embodiments of the present invention, the polypeptide mayinclude the amino acid sequence of SEQ ID NO: 13. In addition, thepolypeptide may be a polypeptide comprising the amino acid sequence ofSEQ ID NO: 13 in which 11, 18, 38, 41, 44, 47, 50, or 53 amino acidresidues are deleted from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 13. Inaddition, the polypeptide may be a polypeptide comprising the amino acidsequence of SEQ ID NO: 13 in which 13, 15, 17, 19, 21, or 23 amino acidresidues are deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 333 of SEQ ID NO: 13.

In addition, the present invention provides a polypeptide represented bythe following Formula (II):N-terminal extension domain-core domain-C-terminal extensiondomain  (II),

in the above Formula (II),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 37;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 39 in which 1 to 41 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 41 in which 1 to 15 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 16 of SEQ ID NO: 41.

The core domain is a polypeptide comprising the amino acid sequence fromposition 154 to position 317 of the full-length Nkx3.2 protein. Thefull-length Nkx3.2 protein may include the amino acid sequence of SEQ IDNO: 7, and the core domain may include the amino acid sequence of SEQ IDNO: 37.

The N-terminal extension domain is a domain bound to the N-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 112 to position 153 of the full-lengthNkx3.2 protein. The N-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 39.

The N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 39, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 39 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 amino acidresidues are deleted from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39. Inembodiments of the present invention, the N-terminal extension domainmay be a polypeptide comprising the amino acid sequence of SEQ ID NO:39, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 39in which 11, 18, 38, or 41 amino acid residues are deleted from theN-terminus to the C-terminal direction, starting from the amino acid atposition 1 of SEQ ID NO: 39.

The C-terminal extension domain is a domain bound to the C-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 318 to position 333 of the full-lengthNkx3.2 protein. The C-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 41.

The C-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 41, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 41 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 16 of SEQ ID NO: 41.

Specifically, the C-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 41 in which 13 or 15amino acid residues are deleted from the C-terminus to the N-terminaldirection, starting from the amino acid at position 16 of SEQ ID NO: 41.

In embodiments of the present invention, the C-terminal extension domainmay be a polypeptide comprising the amino acid sequence of SEQ ID NO:41, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 41in which 3, 6, 9, 13, or 15 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 16 of SEQ ID NO: 41.

The polypeptides represented by the above Formula (I) or (II) arefragments of the Nkx3.2 protein and are not naturally present in livingbodies. Nevertheless, the polypeptides are not easily degraded in vivowhile having the activity comparable to the full-length Nkx3.2 protein,and thus can stay present in a body longer than the full-length Nkx3.2,exhibiting an excellent activity.

The present invention provides polynucleotides encoding the polypeptidesrepresented by the above Formula (I) or (II).

The polynucleotides according to the present invention encodes the coredomain, the N-terminal extension domain, and the C-terminal extensiondomain which may include, respectively, the nucleotide sequences of SEQID NO: 2 or 38, SEQ ID NO: 36 or 40, and SEQ ID NO: 6 or 42.

The polynucleotide may include a polynucleotide that encodes a fragmentobtained by deletion of amino acid residues in the N-terminal extensiondomain and C-terminal extension domain as described above. Here, thepolynucleotide may include a polynucleotide substituted with anothernucleotide sequence that expresses the polypeptide of SEQ ID NO: 1, SEQID NO: 35, or SEQ ID NO: 5.

In addition, the polynucleotide may include a polynucleotide thatencodes a fragment obtained by deletion of amino acid residues in theN-terminal extension domain and C-terminal extension domain as describedabove. Here, the polynucleotide may include a polynucleotide substitutedwith another nucleotide sequence that expresses the polypeptide of SEQID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

The present invention provides expression vectors comprising thepolynucleotides.

The expression vector may be a plasmid vector, a cosmid vector, abacteriophage vector, or a viral vector. The expression vector can beconstructed by a person of ordinary skill in the art, such that thepolynucleotides according to the present invention can be expressed andsecreted therein.

In addition, the present invention provides host cells harboring theexpression vectors.

The host cell is a cell transfected with an expression vector comprisingthe polynucleotide according to the present invention, and may be aprokaryotic cell or a eukaryotic cell. Specifically, the host cell maybe a mammalian cell. The transfection can be carried out using themethods known in the art. Meanwhile, an example of the prokaryotic cellmay be E. coli, and an example of the eukaryotic cell may be yeast. Inaddition, the mammalian cell may be NS/0 myeloma cells, 293 cells,Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (humanamniotic fluid-derived cells), or COS cells.

The present invention provides recombinant viruses comprising thepolynucleotides provided herein.

The virus may be any one selected from the group consisting of anadenovirus, an adeno-associated virus (AAV), a retrovirus, a lentivirus,a herpes simplex virus, and a vaccinia virus. Specifically, the virusmay be an adeno-associated virus (AAV). The adeno-associated virus isnot limited to a specific serotype, and in some embodiments, the AAV maybe any one of AAV1 to AAV9.

Since the adeno-associated virus (AAV) is capable of infectingnon-dividing cells and has an ability to infect various types of cells,the adeno-associated virus is suitably used as a gene delivery system ofthe present invention. Details for preparation and uses of AAV vectorsare described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368.

Typically, the AAV can be produced by co-transfection of a plasmidcomprising a gene sequence of interest which is flanked by two AAVterminal repeats and an expression plasmid comprising a wild-type AAVcoding sequence that does not include the terminal repeats.

In embodiments of the present invention, the present inventors producedNkx3.2 fragments, and found that the fragments are not degraded by Siah1(FIG. 1). The inventors also found that NKx3.2 fragments provided hereininduce the degradation of IκBα through binding to IκBα (FIGS. 2 and 3)and induce transcriptional activation of NF-κB (FIG. 4). In addition,the present inventors found that when the adeno-associated virus thatincludes the polynucleotide encoding the Nkx3.2 fragment is administeredto degenerative arthritis-induced mice, damaged cartilage tissue isrestored (FIGS. 7 and 8). Therefore, the Nkx3.2 fragments of the presentinvention can be effectively used for preventing or treating arthritis.

The present invention provides pharmaceutical compositions forpreventing or treating arthritis, comprising a polypeptide providedherein as an active ingredient. Specifically, the present inventionprovides pharmaceutical compositions for preventing or treatingarthritis, comprising a Nkx3.2 fragment provided herein as an activeingredient.

The Nkx3.2 fragment may be a polypeptide represented by the followingFormula (I):N-terminal extension domain-core domain-C-terminal extensiondomain  (I),

in the above Formula (I),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 1;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 35 in which 1 to 53 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 35; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 5 in which 1 to 23 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

The core domain is a polypeptide comprising the amino acid sequence fromposition 166 to position 309 of the full-length Nkx3.2 protein. Thefull-length Nkx3.2 protein may include the amino acid sequence of SEQ IDNO: 7, and the core domain may include the amino acid sequence of SEQ IDNO: 1.

The N-terminal extension domain is a domain bound to the N-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 112 to position 165 of the full-lengthNkx3.2 protein. The N-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 35.

The N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 35, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 35 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, or 53 amino acid residues are deleted fromthe N-terminus to the C-terminal direction, starting from the amino acidat position 1 of SEQ ID NO: 35. In embodiments of the present invention,the N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 35, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 35 in which 11, 18, 38, 41, 44, 47,50, or 53 amino acid residues are deleted from the N-terminus to theC-terminal direction, starting from the amino acid at position 1 of SEQID NO: 35.

The C-terminal extension domain is a domain bound to a C-terminus of theabove-mentioned core domain, and is a polypeptide comprising the aminoacid sequence from position 310 to position 333 of the full-lengthNkx3.2 protein. The C-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 5.

The C-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 5, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 5 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acidresidues are deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

Specifically, the C-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5, or a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5 in which 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues are deleted fromthe C-terminus to the N-terminal direction, starting from the amino acidat position 24 of SEQ ID NO: 5.

In certain embodiments, the C-terminal extension domain may be apolypeptide comprising the amino acid sequence of SEQ ID NO: 5, or apolypeptide comprising the amino acid sequence of SEQ ID NO: 5 in which13, 15, 17, 19, 21, or 23 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 24 of SEQ ID NO: 5.

Deletion of the amino acid residues may occur in either or both of theN-terminal extension domain and the C-terminal extension domain. In someembodiments, the polypeptide may include the amino acid sequence of SEQID NO: 13, 14, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

The present invention provides a polypeptide comprising the amino acidsequence of SEQ ID NO: 13 or a fragment thereof. The fragment may be apolypeptide comprising the amino acid sequence of SEQ ID NO: 13 in which1 to 53 amino acids are consecutively deleted from the N-terminus to theC-terminal direction, starting from the amino acid at position 1 of SEQID NO: 13. In addition, the fragment may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 13 in which 1 to 23 amino acids areconsecutively deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 333 of SEQ ID NO: 13.

In embodiments of the present invention, the polypeptide may include theamino acid sequence of SEQ ID NO: 13. In addition, the polypeptide maybe a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 inwhich 11, 18, 38, 41, 44, 47, 50, or 53 amino acid residues are deletedfrom the N-terminus to the C-terminal direction, starting from the aminoacid at position 1 of SEQ ID NO: 13. In addition, the polypeptide may bea polypeptide comprising the amino acid sequence of SEQ ID NO: 13 inwhich 13, 15, 17, 19, 21, or 23 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 333 of SEQ ID NO: 13.

In addition, the Nkx3.2 fragment may be a polypeptide represented by thefollowing Formula (II):N-terminal extension domain-core domain-C-terminal extensiondomain  (II),

in the above Formula (II),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 37;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 39 in which 1 to 41 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 41 in which 1 to 15 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 16 of SEQ ID NO: 41.

The core domain is a polypeptide comprising the amino acid sequence fromposition 154 to position 317 of the full-length Nkx3.2 protein. Thefull-length Nkx3.2 protein may include the amino acid sequence of SEQ IDNO: 7, and the core domain may include the amino acid sequence of SEQ IDNO: 37.

The N-terminal extension domain is a domain bound to the N-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 112 to position 153 of the full-lengthNkx3.2 protein. The N-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 39.

The N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 39, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 39 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 amino acidresidues are deleted from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39. Inembodiments of the present invention, the N-terminal extension domainmay be a polypeptide comprising the amino acid sequence of SEQ ID NO:39, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 39in which 11, 18, 38, or 41 amino acid residues are deleted from theN-terminus to the C-terminal direction, starting from the amino acid atposition 1 of SEQ ID NO: 39.

The C-terminal extension domain is a domain bound to the C-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 318 to position 333 of the full-lengthNkx3.2 protein. The C-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 41.

The C-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 41, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 41 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 16 of SEQ ID NO: 41.

Specifically, the C-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 41 in which 13 or 15amino acid residues are deleted from the C-terminus to the N-terminaldirection, starting from the amino acid at position 16 of SEQ ID NO: 41.

In embodiments of the present invention, the C-terminal extension domainmay be a polypeptide comprising the amino acid sequence of SEQ ID NO:41, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 41in which 3, 6, 9, 13, or 15 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 16 of SEQ ID NO: 41.

The polypeptides represented by the above Formula (I) or (II) arefragments of the Nkx3.2 protein and are not naturally present in livingbodies. However, the polypeptide is not easily degraded in vivo whilehaving activity comparable to the full-length Nkx3.2 protein, and thuscan stay present in a body longer than the full-length Nkx3.2,exhibiting an excellent activity.

The Nkx3.2 fragment can be obtained by a host cell transfected with anexpression vector that includes a polynucleotide encoding thepolypeptide represented by (I) or (II).

The polynucleotide encodes the above-mentioned core domain, N-terminalextension domain, and C-terminal extension domain which may include thenucleotide sequences of SEQ ID NO: 2 or 38, SEQ ID NO: 36 or 40, and SEQID NO: 6 or 42, respectively.

The polynucleotide may include a polynucleotide that encodes a fragmentobtained by deletion of amino acid residues in the N-terminal extensiondomain and C-terminal extension domain as described above. Here, thepolynucleotide may include a polynucleotide substituted with anothernucleotide sequence that expresses the polypeptide of SEQ ID NO: 1, SEQID NO: 35, or SEQ ID NO: 5.

In addition, the polynucleotide may include a polynucleotide thatencodes a fragment obtained by deletion of amino acid residues in theN-terminal extension domain and C-terminal extension domain as describedabove. Here, the polynucleotide may include a polynucleotide substitutedwith another nucleotide sequence that expresses the polypeptide of SEQID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

The expression vector may be a plasmid vector, a cosmid vector, abacteriophage vector, or a viral vector. The expression vector can beconstructed by a person of ordinary skill in the art, such that thepolynucleotide according to the present invention can be expressed andsecreted therein.

The host cell is a cell transfected with an expression vector comprisingthe polynucleotide according to the present invention, and may be aprokaryotic cell or a eukaryotic cell. Specifically, the host cell maybe a mammalian cell. The transfection can be carried out by methodsknown in the art. Meanwhile, an example of the prokaryotic cell may beE. coli, and an example of the eukaryotic cell may be yeast. Inaddition, the mammalian cell may be NS/0 myeloma cells, 293 cells,Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (humanamniotic fluid-derived cells), or COS cells.

The arthritis may be any one selected from the group consisting ofosteoarthritis, rheumatoid arthritis, degenerative arthritis, goutyarthritis, juvenile arthritis, senescent arthritis, reactive arthritis,and combinations thereof.

The pharmaceutical composition may include 0.1% to 99% by weight, 1% to90% by weight, and 10% to 80% by weight of the polypeptide according tothe present invention as an active ingredient, relative to the totalweight of the pharmaceutical composition. In addition, thepharmaceutical composition of the present invention may further includeone or more active ingredients which exhibit the same or similarfunction in addition to the above-described active ingredient.

The pharmaceutical composition according to the present invention mayfurther include one or more pharmaceutically acceptable carriers foradministration in addition to the above-described active ingredients.

The dosage of the pharmaceutical composition for preventing or treatingarthritis which includes the Nkx 3.2 fragments as an active ingredientmay be adjusted depending on various factors comprising the type of thedisease, severity of the disease, types and contents of activeingredients and other ingredients included in the pharmaceuticalcomposition, the type of formulation, patient's age, body weight,general health condition, sex, and diet, times of administration, routesof administration, duration of treatment, and drugs simultaneously used.

However, for a desired effect, the dosage of the polypeptide included inthe pharmaceutical composition according to the present invention may be0.0001 to 100 mg/kg. Here, administration may be carried out once a dayor divided into several times.

The present invention provides a pharmaceutical composition forpreventing or treating arthritis, comprising the recombinant virus as anactive ingredient. Specifically, the present invention provides apharmaceutical composition for preventing or treating arthritis,comprising, as an active ingredient, a recombinant virus that includes apolynucleotide encoding the Nkx3.2 fragment.

The polynucleotide loaded on the recombinant virus may encode apolypeptide represented by the following Formula (I):N-terminal extension domain-core domain-C-terminal extensiondomain  (I),

in the above Formula (I),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 1;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 35 in which 1 to 53 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 35; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 5 in which 1 to 23 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

The core domain is a polypeptide comprising the amino acid sequence fromposition 166 to position 309 of the full-length Nkx3.2 protein. Thefull-length Nkx3.2 protein may include the amino acid sequence of SEQ IDNO: 7, and the core domain may include the amino acid sequence of SEQ IDNO: 1.

The N-terminal extension domain is a domain bound to the N-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 112 to position 165 of the full-lengthNkx3.2 protein. The N-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 35.

The N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 35, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 35 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, or 53 amino acid residues are deleted fromthe N-terminus to the C-terminal direction, starting from the amino acidat position 1 of SEQ ID NO: 35. In embodiments of the present invention,the N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 35, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 35 in which 11, 18, 38, 41, 44, 47,50, or 53 amino acid residues are deleted from the N-terminus to theC-terminal direction, starting from the amino acid at position 1 of SEQID NO: 35.

The C-terminal extension domain is a domain bound to the C-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 310 to position 333 of the full-lengthNkx3.2 protein. The C-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 5.

The C-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 5, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 5 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acidresidues are deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 24 of SEQ ID NO: 5.

Specifically, the C-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5, or a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5 in which 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues are deleted fromthe C-terminus to the N-terminal direction, starting from the amino acidat position 24 of SEQ ID NO: 5.

In embodiments of the present invention, the C-terminal extension domainmay be a polypeptide comprising the amino acid sequence of SEQ ID NO: 5,or a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 inwhich 13, 15, 17, 19, 21, or 23 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 24 of SEQ ID NO: 5.

Deletion of the amino acid residues may occur in either or both of theN-terminal extension domain and the C-terminal extension domain. Inembodiments of the present invention, the polypeptide may include theamino acid sequence of SEQ ID NO: 13, 14, 20, 21, 22, 23, 24, 25, 26,27, or 28.

In addition, the polynucleotide loaded on the recombinant virus mayencode a polypeptide represented by the following Formula (II):N-terminal extension domain-core domain-C-terminal extensiondomain  (II),

In the above Formula (II),

the core domain is a polypeptide comprising the amino acid sequence ofSEQ ID NO: 37;

the N-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 39 in which 1 to 41 amino acids areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39; and

the C-terminal extension domain is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 41 in which 1 to 15 amino acids areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid at position 16 of SEQ ID NO: 41.

The core domain refers to a polypeptide comprising the amino acidsequence from position 154 to position 317 of the full-length Nkx3.2protein. The full-length Nkx3.2 protein may include the amino acidsequence of SEQ ID NO: 7, and the core domain may include the amino acidsequence of SEQ ID NO: 37.

The N-terminal extension domain is a domain bound to the N-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 112 to position 153 of the full-lengthNkx3.2 protein. The N-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 39.

The N-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 39, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 39 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 amino acidresidues are deleted from the N-terminus to the C-terminal direction,starting from the amino acid at position 1 of SEQ ID NO: 39. Inembodiments of the present invention, the N-terminal extension domainmay be a polypeptide comprising the amino acid sequence of SEQ ID NO:39, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 39in which 11, 18, 38, or 41 amino acid residues are deleted from theN-terminus to the C-terminal direction, starting from the amino acid atposition 1 of SEQ ID NO: 39.

The C-terminal extension domain is a domain bound to the C-terminus ofthe above-mentioned core domain, and is a polypeptide comprising theamino acid sequence from position 318 to position 333 of the full-lengthNkx3.2 protein. The C-terminal extension domain may include the aminoacid sequence of SEQ ID NO: 41.

The C-terminal extension domain may be a polypeptide comprising theamino acid sequence of SEQ ID NO: 41, or a polypeptide comprising theamino acid sequence of SEQ ID NO: 41 in which 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 amino acid residues are deleted from theC-terminus to the N-terminal direction, starting from the amino acid atposition 16 of SEQ ID NO: 41.

Specifically, the C-terminal extension domain may be a polypeptidecomprising the amino acid sequence of SEQ ID NO: 41 in which 13 or 15amino acid residues are deleted from the C-terminus to the N-terminaldirection, starting from the amino acid at position 16 of SEQ ID NO: 41.

In an embodiment of the present invention, the C-terminal extensiondomain may be a polypeptide comprising the amino acid sequence of SEQ IDNO: 41, or a polypeptide comprising the amino acid sequence of SEQ IDNO: 41 in which 3, 6, 9, 13, or 15 amino acid residues are deleted fromthe C-terminus to the N-terminal direction, starting from the amino acidat position 16 of SEQ ID NO: 41.

The polypeptide represented by the above Formula (I) or (II) is afragment of the Nkx3.2 protein and is not present in vivo. However, thepolypeptide is not easily degraded in vivo while having the sameactivity as the full-length Nkx3.2 protein, and thus is present in abody longer than the full-length Nkx3.2 to exhibit excellent activity.

A recombinant virus that includes a polynucleotide encoding the Nkx3.2fragment can be obtained through a host cell transfected with anexpression vector that includes a polynucleotide encoding thepolypeptide represented by (I) or (II).

The polynucleotide encodes the above-mentioned core domain, N-terminalextension domain, and C-terminal extension domain which may include thenucleotide sequences of SEQ ID NO: 2 or 38, SEQ ID NO: 36 or 40, and SEQID NO: 6 or 42, respectively.

The polynucleotide may include a polynucleotide that encodes a fragmentobtained by deletion of amino acid residues in the N-terminal extensiondomain and C-terminal extension domain as described above. Here, thepolynucleotide may include a polynucleotide substituted with anothernucleotide sequence that expresses the polypeptide of SEQ ID NO: 1, SEQID NO: 35, or SEQ ID NO: 5.

In addition, the polynucleotide may include a polynucleotide thatencodes a fragment obtained by deletion of amino acid residues in theN-terminal extension domain and C-terminal extension domain as describedabove. Here, the polynucleotide may include a polynucleotide substitutedwith another nucleotide sequence that expresses the polypeptide of SEQID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

The polynucleotide may include a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 13, 14, 20, 21, 22, 23, 24, 25, 26, 27, or28.

The virus may be any one selected from the group consisting of anadenovirus, an adeno-associated virus (AAV), a retrovirus, a lentivirus,a herpes simplex virus, and a vaccinia virus. Specifically, the virusmay be an adeno-associated virus (AAV). The adeno-associated virus isnot limited to a specific serotype, and preferably, may be any one ofAAV1 to AAV9.

Since the adeno-associated virus (AAV) is capable of infectingnon-dividing cells and has an ability to infect various types of cells,the adeno-associated virus is suitably used as a gene delivery system ofthe present invention. Details for preparation and uses of AAV vectorsare described in U.S. Pat. Nos. 5,139,941 and 4,797,368.

Typically, the AAV can be produced by co-transfection of a plasmidcomprising a gene sequence of interest which is flanked by two AAVterminal repeats and an expression plasmid comprising a wild-type AAVcoding sequence which does not have the terminal repeats.

The arthritis may be any one selected from the group consisting ofosteoarthritis, rheumatoid arthritis, degenerative arthritis, goutyarthritis, juvenile arthritis, senescent arthritis, reactive arthritis,and combinations thereof.

The pharmaceutical composition according to the present invention mayfurther include one or more pharmaceutically acceptable carriers foradministration in addition to the above-described active ingredients.

The dosage of the pharmaceutical composition for preventing or treatingarthritis which includes, as an active ingredient, a recombinant virusthat includes a polynucleotide encoding the Nkx 3.2 fragment may beadjusted depending on various factors including the type of the disease,severity of the disease, types and contents of active ingredients andother ingredients included in the pharmaceutical composition, the typeof formulation, patient's age, body weight, general health condition,sex, and diet, times of administration, routes of administration,duration of treatment, and drugs simultaneously used.

However, for a desired effect, the recombinant virus included in thepharmaceutical composition according to the present invention may beadministered in an amount of 1.0×10⁵ to 1.0×10¹⁵ viral genome per day inthe case of adults. Specifically, the dosage of the pharmaceuticalcomposition of the present invention may be such that administration iscarried out in an amount of 1.0×10⁵ to 1.0×10¹⁵, 1.0×10⁷ to 1.0×10¹³,1.0×10⁸ to 1.0×10¹², or 1.0×10⁹ to 1.0×10¹⁰ per day in the case ofadults.

The present invention provides a method of preventing or treatingarthritis, comprising the step of administering the pharmaceuticalcomposition to a subject in need thereof. Specifically, the presentinvention provides a method of preventing or treating arthritis,comprising the step of administering, to a subject in need thereof, apharmaceutical composition for preventing or treating arthritis, whichincludes, as an active ingredient, the Nkx3.2 fragment or a recombinantvirus that includes a polynucleotide encoding the Nkx 3.2 fragment.

The subject may be a mammal, in particular, a human. The route ofadministration can be appropriately selected by a person skilled in theart in consideration of an administration method, volume and viscosityof body fluid, and the like. Specifically, the pharmaceuticalcomposition may be intra-articularly administered.

The pharmaceutical composition may be intra-articularly administered. Asused herein, the term “intra-articularly” means that administration iscarried out via a lumen enclosed by an articular capsule, which is a gapbetween bones in a joint. There are various methods to carry outintra-articular administration. For example, there is a method in whicha patient is asked to bend one knee 90 degrees in a state of lying downat a posture looking at the ceiling, and a syringe is intra-articularlyinserted. In this posture, the inside and the outside joint boundariescan be relatively easily distinguished by hand. Injection can be carriedout at either or both of the inside and the outside joint boundaries,and is mostly carried out toward the inside joint boundary. In addition,there is also a method of carrying out injection at a posture where aknee is stretched. For both postures, when the syringe is correctlyinserted into a predetermined injection site, the injection solution maybe injected with little resistance. However, when, at the time ofpressing a syringe, drugs do not enter well, and a sense of resistanceis recognized or a patient complains of severe pain, the injection siteof the syringe should be adjusted.

The present invention provides a method of producing an Nkx3.2 fragmentwith increased stability in a body, comprising the step of deleting anyone region of a polypeptide comprising the amino acid sequence of SEQ IDNO: 7, which is selected from the group consisting of an N-terminalregion and a C-terminal region, and a combination thereof.

Deletion of the N-terminal region may be such that 1 to 165 amino acidsare consecutively deleted from the N-terminus to the C-terminaldirection, starting from the amino acid at position 1 of SEQ ID NO: 7.Specifically, the deletion may be such that 1 to 53 amino acids areconsecutively deleted from the N-terminus to the C-terminal direction,starting from the amino acid at position 112 of SEQ ID NO: 7. In anembodiment of the present invention, the deletion of the N-terminalregion may be such that 11, 18, 38, 41, 44, 47, 50, or 53 amino acidresidues are deleted from the N-terminus to the C-terminal direction,starting from the amino acid at position 112 of SEQ ID NO: 7.

Deletion of the C-terminal region may be such that 1 to 23 amino acidsare consecutively deleted from the C-terminus to the N-terminaldirection, starting from the amino acid at position 333 of SEQ ID NO: 7.In an embodiment of the present invention, the deletion of theC-terminal region may be such that 13, 15, 17, 19, 21, or 23 amino acidresidues are deleted from the C-terminus to the N-terminal direction,starting from the amino acid at position 333 of SEQ ID NO: 7.

The deletion of the amino acid residue may occur at either or both ofthe N-terminal region and the C-terminal region. In an embodiment of thepresent invention, the Nkx3.2 fragment may include the amino acidsequence of SEQ ID NO: 13, 14, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

The deletion of the amino acid residues can be carried out with anappropriate method by a person skilled in the art. The Nkx3.2 fragmentswith increased stability in a body produced by the above method are noteasily degraded in vivo by Siah1, and thus may be present in a bodylonger than the wild type Nkx3.2 protein.

MODES FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail by thefollowing examples. However, the following examples are intended merelyto illustrate the present invention, and the present invention is notlimited thereto.

Example 1. Construction of Vectors Expressing Nkx3.2 Fragments

In order to obtain variants which are resistant to proteolysis mediatedby Siah1, vectors expressing Nkx3.2 fragments were constructed by thefollowing method.

Specifically, the Nkx3.2 gene having the nucleotide sequence representedby SEQ ID NO: 8 was used as a template and amplified using a Lamp PfuDNA polymerase (Cat. #LP116-250, BIOFACT Co., Ltd., Korea) according tothe manufacturer's protocol. Each of the amplified PCR products wascleaved with restriction enzymes EcoRI (Cat. #FD0274, Thermo FisherScientific Inc., USA), and XhoI (Cat. #FD0694, Thermo Fisher ScientificInc., USA) or XbaI (Cat. #FD0684, Thermo Fisher Scientific Inc., USA),and respectively, inserted into a pCS expression vector (Addgene Cat#17095) using a T4 ligase (Cat. #EL0011, Thermo Fisher Scientific Inc.,USA).

Consequently, expression vectors expressing 20 kinds of Nkx3.2 fragmentswere constructed as shown in Table 1 below.

TABLE 1 Name Feature SEQ ID NO Nkx3.2 (1-333) Full-length Nkx3.2 SEQ IDNO: 7 Nkx3.2 (1-320) Nkx3.2 fragment containing 1st to 320th amino acidsSEQ ID NO: 9 Nkx3.2 (1-307) Nkx3.2 fragment containing 1st to 307thamino acids SEQ ID NO: 10 Nkx3.2 (42-333) Nkx3.2 fragment containing42nd to 333rd amino acids SEQ ID NO: 11 Nkx3.2 (99-333) Nkx3.2 fragmentcontaining 99th to 333rd amino acids SEQ ID NO: 12 Nkx3.2 (112-333)Nkx3.2 fragment containing 112th to 333rd amino acids SEQ ID NO: 13Nkx3.2 (123-333) Nkx3.2 fragment containing 123rd 333rd amino acids SEQID NO: 14 Nkx3.2 (99-330) Nkx3.2 fragment containing 99th to 330th aminoacids SEQ ID NO: 15 Nkx3.2 (99-327) Nkx3.2 fragment containing 99th to327th amino acids SEQ ID NO: 16 Nkx3.2 (99-320) Nkx3.2 fragmentcontaining 99th to 320th amino acids SEQ ID NO: 17 Nkx3.2 (105-327)Nkx3.2 fragment containing 105th to 327th amino acids SEQ ID NO: 18Nkx3.2 (110-324) Nkx3.2 fragment containing 110th to 324th amino acidsSEQ ID NO: 19 Nkx3.2 (112-320) Nkx3.2 fragment containing 112th to 320thamino acids SEQ ID NO: 20 Nkx3.2 (123-320) Nkx3.2 fragment containing123rd 320th amino acids SEQ ID NO: 21 Nkx3.2 (130-320) Nkx3.2 fragmentcontaining 130th to 320th amino acids SEQ ID NO: 22 Nkx3.2 (150-320)Nkx3.2 fragment containing 150th to 320th amino acids SEQ ID NO: 23Nkx3.2 (153-318) Nkx3.2 fragment containing 153rd 318th amino acids SEQID NO: 24 Nkx3.2 (156-316) Nkx3.2 fragment containing 156th to 316thamino acids SEQ ID NO: 25 Nkx3.2 (159-314) Nkx3.2 fragment containing159th to 314th amino acids SEQ ID NO: 26 Nkx3.2 (162-312) Nkx3.2fragment containing 162nd to 312th amino acids SEQ ID NO: 27 Nkx3.2(165-310) Nkx3.2 fragment containing 165th to 310th amino acids SEQ IDNO: 28

Example 2. Selection of Nkx3.2 Fragments Resistant to ProteolysisMediated by Siah1

Using the expression vectors expressing the Nkx3.2 fragments asconstructed in Example 1, Nkx3.2 fragments which are not degraded bySiah1 were selected by the following method.

First, Siah1 (SEQ ID NO: 29; GenBank Accession No. AAH35562.1) wasamplified by PCR in the same condition and method as described inExample 1, and the amplified PCR product was cleaved with EcoRI andNcoI. The resulting product was inserted into a pCS 3HA expressionvector (Addgene plasmid #17095, a vector with a 3-HA epitope taginserted between EcoRI and ClaI sites of pCS2P+), which had been cleavedwith the same restriction enzymes and includes a tag in which the humaninfluenza hemagglutinin (HA) amino acid sequence (SEQ ID NO: 33;YPYDVPDYA) is repeated three times, to construct an expression vectorexpressing Siah1.

Meanwhile, 293T kidney cell line (Cat. #CRL-3216, ATCC, USA) wascultured in a DMEM (Dulbecco's modified Eagle's medium) mediumsupplemented with 10% (v/v) fetal bovine serum (FBS) at a condition of37° C. and 5% CO₂. The prepared cells were dispensed on a 60×15 mm cellculture plate so that the number of cells was 5×10⁵. The cells weretransiently transfected using 2 μg of the expression vector expressingNkx3.2, and 4 μg of each of the expression vectors expressing the Nkx3.2fragments, respectively, together with 2 μg of the expression vectorexpressing Siah1. The transfection was carried out using VivaMagic (Cat.#VM001, VIVAGEN CO., LTD., Korea) according to the manufacturer'sprotocol.

The entire protein was isolated from the transfected cells andquantitated using a Bio-Rad Laboratories protein kit (Cat. #500-0116,Bio-Rad Laboratories, Inc., USA). Then, western blotting for each ofNkx3.2, Siah1, and β-actin was carried out by a conventional method.Here, an anti-Nkx3.2 antibody (Cat. #Ab83288, Abcam, Great Britain), ananti-HA antibody (Cat. #11583816001, Roche, Switzerland), an anti-Mycantibody (Cat. #11667149001, Roche, Switzerland), and an anti-β-actinantibody (Cat. #LF-PA0207, AbFrontier, Korea) were diluted at 1:1,000,1:5,000, 1:5,000, and 1:5,000, respectively, in a TBST buffer containing3% (v/v) bovine serum albumin (BSA), and used. As a result, photographsof western blotting bands are illustrated in FIG. 1, which is summarizedin Table 2 below.

TABLE 2 Name SEQ ID NO Degradation by Siah1 Nkx3.2(1-333) SEQ ID NO: 7+++ ∘ Nkx3.2(1-320) SEQ ID NO: 9 + x Nkx3.2(1-307) SEQ ID NO: 10 − xNkx3.2(42-333) SEQ ID NO: 11 +++ ∘ Nkx3.2(99-333) SEQ ID NO: 12 ++ xNkx3.2(112-333) SEQ ID NO: 13 ++ x Nkx3.2(123-333) SEQ ID NO: 14 − xNkx3.2(99-330) SEQ ID NO: 15 +++ ∘ Nkx3.2(99-327) SEQ ID NO: 16 +++ ∘Nkx3.2(99-320) SEQ ID NO: 17 + x Nkx3.2(105-327) SEQ ID NO: 18 ++ ∘Nkx3.2(110-324) SEQ ID NO: 19 +++ ∘ Nkx3.2(112-320) SEQ ID NO: 20 − xNkx3.2(123-320) SEQ ID NO: 21 − x Nkx3.2(130-320) SEQ ID NO: 22 − xNkx3.2(150-320) SEQ ID NO: 23 − x Nkx3.2(153-318) SEQ ID NO: 24 − xNkx3.2(156-316) SEQ ID NO: 25 ++ x Nkx3.2(159-314) SEQ ID NO: 26 ++ xNkx3.2(162-312) SEQ ID NO: 27 ++ x Nkx3.2(165-310) SEQ ID NO: 28 ++ x

As illustrated in FIG. 1 and shown in Table 2, unlike the full-lengthNkx3.2 (1-333), a degradation of Nkx3.2 protein by Siah1 did not occurin the fragments Nkx3.2 (1-320), Nkx3.2 (1-307), Nkx 3.2 (123-333), Nkx3.2 (99-320), Nkx 3.2 (112-320), Nkx 3.2 (123-320), Nkx 3.2 (130-320),Nkx3.2 (150-320), and Nkx3.2 (153-318).

Example 3. Identifying Whether Nkx3.2 Fragments Bind to IκBα

Nkx3.2 induces NF-κB activation through binding to IκBα. Thus,immunoprecipitation was used to identify whether the fragments Nkx 3.2(112-320), Nkx 3.2 (123-320), Nkx 3.2 (130-320), Nkx 3.2 (150-320), andNkx 3.2 (153-318) bind to IκBα.

First, IκBα (SEQ ID NO: 31; GenBank Accession No. CAB65556) wasamplified by PCR in the same condition and method as described inExample 1, and the amplified PCR product was cleaved with EcoRI andXbaI. The resulting product was inserted into a pCS 6Myc expressionvector (Addgene plasmid #17095, a vector with 6-Myc epitope tag insertedbetween EcoRI and ClaI sites of pCS2P+), which had been cleaved with thesame restriction enzymes and includes a tag in which the Myc amino acidsequence (SEQ ID NO: 34: EQKLISEEDL) is repeated six times, to constructan expression vector expressing IκBα.

Then, 293T kidney cell line was transfected in the same condition andmethod as described in Example 2 using 8 μg of the expression vectorexpressing Nkx3.2 (1-333) and each of the expression vectors expressingthe fragments, respectively, as produced in Example 1, together with anequal amount of the expression vector expressing IκBα. Here, in order toprevent the IκBα protein from being degraded by the Nkx3.2 protein,MG132 (Cat. #474790, Merck Millipore, Germany), which is aproteasome-degradation suppressor, was added at a concentration of 20μM. After 6 hours, the entire protein was isolated from the cells, andimmunoprecipitation was carried out by a conventional method using ananti-Myc antibody that recognizes the Myc with which IκBα is labeled.Then, western blotting was carried out using the antibodies as describedabove. Photographs of the obtained results are illustrated in FIG. 2.

As illustrated in FIG. 2, similar to the full-length Nkx3.2 (1-333), thebands were formed for the fragments Nkx 3.2 (112-320), Nkx 3.2(123-320), Nkx 3.2 (130-320), Nkx 3.2 (150-320), and Nkx 3.2 (153-318).Therefore, the Nkx3.2 fragments were identified to have the function ofbinding to IκBα to form a complex, which is necessarily required foractivation of NF-κB.

Example 4. Identifying Whether IκBα Protein is Degraded by Nkx3.2Fragments

Nkx3.2 binds to IκBα, and thus promotes ubiquitination and degradationof IκBα by proteasome. Accordingly, western blotting was carried out toidentify whether the fragments Nkx3.2 (112-320), Nkx3.2 (123-320),Nkx3.2 (130-320), Nkx3.2 (150-320), and Nkx3.2 (153-318) maintain suchactivity.

Meanwhile, ATDC5 cartilage cell line (Cat. #RCB0565, Riken, Japan) wascultured in a DMEM/F12 (Dulbecco's modified Eagle's medium: NutrientMixture F-12) medium supplemented with 10% (v/v) fetal bovine serum at acondition of 37° C. and 5% CO₂. The prepared cells were dispensed on a90×20 mm cell culture plate so that the number of cells was 5×10⁵. Thecells were transiently transfected using 4 μg of the expression vectorexpressing Nkx3.2 (1-333) and 8 μg of each of the expression vectorsexpressing the Nkx3.2 fragments, respectively, together with 1 μg of theexpression vector expressing IκBα. The transfection was carried outusing VivaMagic (Cat. #VM001, VIVAGEN CO., LTD., Korea) according to themanufacturer's protocol.

A subsequent process was such that western blotting was carried out inthe same condition and method as described in Example 2, and photographsof the obtained results are illustrated in FIG. 3.

As shown in FIG. 3, bands with an intensity similar to the full-lengthNkx3.2 (1-333) were formed for the fragments Nkx 3.2 (112-320), Nkx 3.2(123-320), Nkx 3.2 (130-320), Nkx 3.2 (150-320), and Nkx3.2 (153-318).Hence, the Nkx3.2 fragments are identified to induce proteolysis of IκBαat the same level as the full-length Nkx3.2.

Example 5. Identifying Whether Transcriptional Function of NF-κB isActivated by Nkx3.2 Fragments

Nkx3.2 suppresses cell death of chondrocytes by inducing NF-κBactivation. Thus, in order to measure the NF-κB activation by Nkx3.2fragments, a polynucleotide sequence in which the NF-κB-specific DNAbinding site (SEQ ID NO: 35: GGGAATTTCC) is repeated four times wasinserted into a pGL3-Basic vector (Cat. #E1751, Promega, USA) using MluIand XhoI to construct a 4×-κB-Luc expression vector. Further, theexpression vector was used to measure activation of transcriptionalfunction of NF-κB by Nkx3.2 by analyzing the luciferase activity.

First, 293T kidney cell line was transiently transfected using,respectively, 200 ng of the expression vector expressing the full-lengthNkx3.2 (1-333) and each of the expression vectors expressing the Nkx3.2fragments, 100 ng of the 4×-κB-Luc expression vector, and 20 ng of apRL-TK expression vector (Cat. #E2241, Promega, USA).

Transfection was carried out using VivaMagic according to themanufacturer's protocol. After 24 hours, the luciferase assay wascarried out using the Dual-Luciferase Reporter Assay System (Cat.#E1910, Promega, USA) according to the manufacturer's protocol.

Specifically, a culture of the transfected 293T kidney cell line wasremoved and washed with 1×PBS. 150 μl of 1× passive lysis buffer (PLB)was added thereto, and the cells were lysed at room temperature for 15minutes. To 10 μl of the cell lysate, 50 μl of LAR II was added and theresultant was allowed to react. Then, the firefly luciferase activitywas measured. To this, 50 μl of Stop & Glo was added and Renillaluciferase activity was measured. In the experimental results, for eachsample, the Renilla luciferase activity was normalized to the fireflyluciferase activity, and an average of percentages therefor isillustrated in FIG. 4.

As shown in FIG. 4, when the luciferase activity in the cellstransfected with only the pCS2 vector as a negative control is set as 1,not only the full-length Nkx3.2 (1-333) but also the fragments Nkx3.2(112-320), NKx3.2 (123-320), Nkx3.2 (130-320), Nkx3.2 (150-320), andNkx3.2 (153-318) exhibited significantly increased luciferase activity.Thus, the Nkx3.2 fragments are identified to possess the function ofactivating the transcriptional function of NF-κB at the level similar tothe full-length Nkx3.2.

Example 6: Identification of the Improved Therapeutic Efficacy of Nkx3.2Fragments Against Degenerative Arthritis

Through the above-described in vitro experiments, functional superiorityof the Nkx3.2 fragments as compared with the full-length Nkx3.2 wasidentified. Accordingly, in order to identify the improved in vivofunction of the Nkx3.2 fragments, the therapeutic efficacy of the Nkx3.2fragment (123-320) and the full-length Nkx3.2 (1-333) againstdegenerative arthritis was compared and analyzed. For this purpose, amouse model in which degenerative arthritis was induced through asurgical procedure called destabilization of medial meniscus (DMM) wasselected. A process for carrying out the experiment is schematicallyillustrated in FIG. 6.

Specifically, the medial meniscus ligament in the knee tissue was cut toinduce structural destabilization of the medial meniscus, and thus thefemoral cartilage and the tibia cartilage were caused to collide againsteach other, so that cartilage damage was induced, thereby inducingdegenerative arthritis. For a control, an animal group for which theouter skin and the inner skin of the knee were dissected and sutured bya mock surgery was used. The animal group for which degenerativearthritis were induced and the control for which the mock surgery wereperformed were grown for 8 weeks. Then, an adeno-associated virus (AAV)expressing the Nkx3.2 fragment (123-320) or the full-length Nkx3.2(1-333), or an empty vector AAV was intra-articularly injected into thecorresponding knee, and the animal groups were grown for 4 weeks. Aprogression degree of degenerative arthritis was analyzed byhistopathological analysis.

For the histopathological analysis, a safranin-O staining method wasemployed. Safranin-O is a cationic compound stain and effectivelyadheres to an anionic group of cartilage heparan sulfate proteoglycan sothat red color is exhibited. A reddish dark-stained area is evaluated tobe cartilage tissue in a healthy condition. Conversely, a part thatexhibits weak or no safranin-O staining, and a part with damaged tissueare interpreted as lesions in which the pathology of degenerativearthritis has progressed.

As shown in FIG. 7, in the case of the control group in which the emptyvector AAV is intra-articularly injected, an extremely severe cartilagedamage and degeneration phenomenon were observed regardless of theamount of viral particles administered.

In the case of the comparison group in which AAV expressing thefull-length Nkx3.2 (1-333) is intra-articularly injected, significanttherapeutic efficacy against degenerative arthritis was observed only inthe AAV-administered group at 1×10¹⁰.

On the contrary, in the case of the experimental group in which AAVexpressing the NKx3.2 fragment (123-320) is intra-articularly injected,a superior therapeutic effect against degenerative arthritis wasexhibited from the AAV-administered group at 1.25×10⁹, which is thelowest dose. That is, in the case of the Nkx3.2 fragment (123-320), thetherapeutic efficacy against degenerative arthritis is identified to beimproved by at least 10 times, compared with the full-length Nkx3.2(1-333).

All data obtained through the histopathological analysis werequantitatively evaluated, and the results are graphically illustrated inFIG. 8. The number of animals analyzed in each experimental group was 3,and severity of degenerative arthritis was evaluated on a scale of 0 to5. From the results, a bar graph was prepared. Mean with SEM wasindicated by an error bar. Virus particle doses A, B, C, and D were1.25×10⁹, 2.5×10⁹, 5×10⁹, and 1×10¹⁰, respectively, in this increasingorder.

As shown in FIG. 8, in the case of the control in which the empty vectorAAV is intra-articularly injected, a high score of 4.5 to 5 wasevaluated regardless of the amount of viral particles administered. Inaddition, in the comparison group in which AAV expressing thefull-length Nkx3.2 (1-333) is intra-articularly injected, a low score of1.5 was evaluated only in the AAV-administered group at 1×10¹⁰. On thecontrary, in the case of the experimental group in which AAV expressingthe NKx3.2 fragment (123-320) is intra-articularly injected, anextremely low score of 1 or less was evaluated starting from theAAV-administered group at 1.25×10⁹, which is the lowest dose. Namely,the Nkx3.2 fragment (123-320) is identified to have superior therapeuticefficacy against degenerative arthritis, compared with the full-lengthNkx3.2 (1-333).

What is claimed is:
 1. A vector comprising a polynucleotide encoding apolypeptide consisting of the following Formula (I): N-terminalextension domain-core domain-C-terminal extension domain (I) wherein thecore domain is a polypeptide having the amino acid sequence of SEQ IDNO: 1; the N-terminal extension domain is a polypeptide having the aminoacid sequence of SEQ ID NO: 3 in which 1 to 42 amino acid residues areconsecutively deletable from the N-terminus to the C-terminal direction,starting from the amino acid residue at position 1 of SEQ ID NO: 3; andthe C-terminal extension domain is a polypeptide having the amino acidsequence of SEQ ID NO: 5 in which 1 to 23 amino acid residues areconsecutively deletable from the C-terminus to the N-terminal direction,starting from the amino acid residue at position 24 of SEQ ID NO:
 5. 2.A pharmaceutical composition comprising the vector of claim 1 and apharmaceutically acceptable carrier.
 3. A recombinant virus comprisingthe polynucleotide according to claim
 1. 4. A host cell infected withthe recombinant virus of claim
 3. 5. A pharmaceutical compositioncomprising the recombinant virus of claim 3 and a pharmaceuticallyacceptable carrier.
 6. The recombinant virus according to claim 3,wherein the virus is selected from the group consisting of anadenovirus, an adeno-associated virus (AAV), a retrovirus, a lentivirus,a herpes simplex virus, and a vaccinia virus.
 7. A vector comprising apolynucleotide encoding a polypeptide selected from the group consistingof SEQ ID NO: 21, 13, 14, 20, 22, 23, 24, 25, 26, 27, or
 28. 8. Apharmaceutical composition comprising the vector of claim 7 and apharmaceutically acceptable carrier.
 9. A recombinant virus comprisingthe polynucleotide according to claim
 7. 10. A pharmaceuticalcomposition comprising the recombinant virus of claim 9 and apharmaceutically acceptable carrier.
 11. A host cell infected with therecombinant virus of claim
 9. 12. The recombinant virus according toclaim 9, wherein the virus is selected from the group consisting of anadenovirus, an adeno-associated virus (AAV), a retrovirus, a lentivirus,a herpes simplex virus, and a vaccinia virus.
 13. A vector comprising apolynucleotide encoding a polypeptide consisting of SEQ ID NO: 12 or 17.14. A pharmaceutical composition comprising the vector of claim 13 and apharmaceutically acceptable carrier.
 15. A recombinant virus comprisingthe polynucleotide according to claim
 13. 16. A pharmaceuticalcomposition comprising the recombinant virus of claim 15 and apharmaceutically acceptable carrier.
 17. A host cell infected with therecombinant virus of claim
 15. 18. The recombinant virus according toclaim 15, wherein the virus is selected from the group consisting of anadenovirus, an adeno-associated virus (AAV), a retrovirus, a lentivirus,a herpes simplex virus, and a vaccinia virus.